Heparin and heparan sulfates (H/HS) are a class of highly sulfated glycoamino-polysaccharide biopolymers found in cells of the various tissues and comprise modulatory receptor systems throughout the body. They have a large diversity in sugar modifications and in sulfate density (aninonic density) along the polysaccharide chains. This structural diversity provides H/HS with a sufficiently large number of unique sequences to account for its known ability to modulate precisely the functions of many diverse proteins and biological systems in normal and disease processes. e.g., cell growth, inflamation, development, blood coagulation, and infections by viruses and other human pathogens. Due to the high degree of similarity of physicochemical properties of H/HS chains carrying these specific sequences and absent the given specific ligands, libraries of the unique H/HS oligoS have not been readily obtainable for research. We developed a macro combinatorial type of heparin-mimetic family (library) based on the structure-function model for the antithrombin-dependent anticoagulant heparin (Rosenberg and coworkers). For this we selected a chemically sulfated natural xylan German phamaceutical comprised of S-oligoS that mimicked unfractionated heparin in almost all its known biological actions, at similar uM concentrations, to obtain a pilot heparin-mimetic family of S-OligoS HD 01315-01-08-10. This enabled us to demonstrate for the first time that the in vitro inhibitions of HIV-1 cytotpathology and syncytium-formation were 1) each governed by a degree of structural specificity and 2) each separability from the anticoagulant S-oligoS of the pharmaceutical and from each other, which are two essential indicators of their usefulness for further drug development AL Stone, et al 1998 Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of Human Immunodeiciency Virus-1 in vitro Glycoconjugate Journal 15:697-71. We devised enlarged procedures to enable the clinical preparation of a highly active HIV-1 virus fusion inhibitor free of anti-thrombin toxicity (PK II, SOLIS) AL Stone and James McMahon, et al:[unreadable] SUMMARY OF PROPERTIES OF PK II [unreadable] *IC 50 vs cell-killing (formazan assay) -- 0.20-0.3 ug/ml; *IC 50 vs virus-cell fusion -- 0.05-0.1 ug/ml; inhib. adherence CD4 cell to gp120 (concentration-dependent); anticoagulation vs thrombin -- less than 0.05-0.3 HU/mg; endotoxin content FDA LAL Assay -- less than 0.06 eu. *N.B.: IC 50 may be 7-10 x greater in a commercial lab; [unreadable] [unreadable] SOLIS: POTENTIAL HIV-1 VIRAL ENTRY INHIBITOR: [unreadable] HIV-infection in Americans is appx. one million and rising, 50 times that worldwide. With no cure nor fully effective vaccine, and the successful long term control of viral load and deadly effects in developed countries threatened by toxicity, multi-drug resistant strains, and reliance on mostly protease and reverse transcriptase inhibitors (drugs approved by the FDA plus only one virus entry inhibitor), broad consensus counsels that inhibitors against viral components that have other functions in the HIV-1 attack are critically needed. We are working on completing a clinical preparation of SOLIS for formulation in a small Phase I i.v.-administration safety trial. SOLIS would be an adjunct drug selected from the family library as an inhibitor of binding and entry of virus into target CD4 cells (See Tabe above). Mechanism of Action: Heparin has long been known to affect conformation of proteins as a means of modulating their biological function (e.g., AL Stone RD Rosenberg et al 1982 PNAS 79:7190, AL Stone P Epstein 1977 BBA 497:298). Thus, inhibition of the conformational changes which are believed to be associated with the formation of the immediate fusigenic structure of gp41 required for fusion progression, is a possible mechanism of action of SOLIS. It has long been established that orally administred S-oligoS are very poorly absorbed, necessitating an i.v. route of administration and consequently a relatively large amount of drug. In addition, clinical studies using this sulfated xylan pharmaceutical can be flawed by the use of indirect means to assess blood levels of the drug during i.v. trials. [unreadable] Currently: We have completed the preparation of Stage II product, SOLIS, from the first lot of SP54 pharmaceutical starting material, with appx.1.6 g conjoined plus aaaadditions on hand. We are negotiatingg a Confiiiidentiality Agreement as well as a materials transfer with the pharmaceutical company to facilitate scientific progress and to obtain SP54 rapidly for preparation of additional SOLIS for the Phase I trial. We completed the pilot study of a US FDA licensed drug derived from SP54 as a potential alternate starting material for preparation of SOLIS HD001315-13. Final evaluation indicated that differences in the physicochemical properties of the mixture of S-oligoS in the derived US product from those of SP54 caused differences and difficulties in its fractionation and the isolation of Components as compared with the properties of the SP54 library and SOLIS, so that it is not well suited to be an alternate source. STUDIES ON COMBINATION THERAPY: Currently, Cp11 samples on hand were combined for studies on whether the addition of increasing amounts of Cp11 (relatively inactive vs HIV-1) to the standard Pk II doses in the formazan cytotoxicity protection assay would effect the capacity of Pk II to protect cells for report see Z01 HD008733-05. ENLARGEMENT OF LIBRARY: Further enlargement of our library in the preparation of Cp11 is ongoing. Preparation of CpC (smallest mass component retaining high activity against cytopatholgical effects of HIV-1 in vitro.) is ongoing. [unreadable] CpC will be used to prepare derivatives (conjugates) using selected polypeptides for various immunological approahes as well as for structure-function studies (See below) on SOLIS ; 1) to obtain a potent immunogen that would serve to produce a specific antibody reagent for direct measurement of SOLIS concentraion in the blood after i.v. administration; 2) to obtain antibodies against putative viral fusion-specific conformations. VIRUS LIGANDS AND BIFUNCTIONAL SOLIS PROBE: Our proposal See previous HD 00 1315- reports to identify and isolate putative H/HS ligand(s) under conditions of HIV-1 tissue culture infection in the presence of a bifunctional SOLIS probe, and to achieve chemical bonding to endogenous viral/cell protein structures during virus attack and subsequently isolate the putative endogenous protein-probe is ongoing. Such ligand(s) could be used to obtain endogenous receptors and/or might be protective antigens. DERIVATIZATION OF S-OLIGOS: S-oligoS of interest are derivatized following our procedures for momo-derivatizing library S-oligoS at the reducing end, at pH close to 3.0 (See earlier HD 00-1315 reports). Our proposal to select particular synthetic polypeptides for linkage based on our speculation of th mode of action of SOLIS for producing specific immunologic reagents and immunogens is ongoing.[unreadable] [unreadable] STRUCTURE-FUNCTION (SF) RELATIONS: : Early on we discovered that S-oligoS remaining after chemical sulfation of the native xylan comprised a family of oligomers that range in mass from about 20 to less than 2 K on average. These contain decreasing numbers of a putative -D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 (tetrasacchaide) motif and varying amounts of axial sulfates (alterrnative chair conformation).(See previous HD 00 1315 reports). Currently we are preparing Cp II and Cp 8b for advanced FTIR spectral analysis and HMQC correlation spectra to determine the relation of alternative chair structure in the inhibition of HIV-1 gp120 and gp41.